Compounds and methods for treating cancer, viral infections, and allergic conditions

This invention was made with government support under CA224832 awarded by the National Institutes of Health. The government has certain rights in the invention.

The present invention generally relates to compounds that are useful for inhibiting one or more proteases including various serine proteases such as Hepatocyte Growth Factor Activator (HGFA), matriptase, and hepsin. The compounds are also useful as inhibitors of kallikrein related peptidase 5 (KLK5) and TMPRSS2 (required for the host cellular entry of SARS-CoV-2). The present invention also relates to various methods of using the inhibitor compounds to treat and/or prevent infections, including those caused by coronaviruses and influenza viruses, conditions associated with KLK5 activity (e.g. inflammation), various other malignancies, pre-malignant conditions, and/or cancer.

Proteases, also known as proteinases, peptidases, or proteolytic enzymes, are enzymes that process proteins by hydrolyzing peptide bonds between amino acid residues. It is known that proteases regulate numerous physiological processes which enable or stimulate the growth, proliferation, transformation, motility, survival, and metastasis of tumor cells. Metastasis involves the proteolytic degradation of the extracellular matrix proteins (e.g. collagen) surrounding the tumor cells by proteases, which enables the invasion of tumor cells metastasizing from the primary tumors into the surrounding tissue and the lymph system or the blood system. Theses degradation proteases include matrix metalloproteases (MMPs) and cysteine proteases including various subfamily members of the cathepsins. Proteases are also involved in the activation of growth factors, cytokines, and other proteins that stimulate the growth, proliferation, motility, and survival of cancer cells, thus enabling tumors to develop and expand in size. These include multiple members of trypsin-like S1 serine protease such as HGFA, KLK5 and of the subfamily of serine proteases called type II transmembrane serine proteases (TTSPs) such as matriptase, hepsin, and TMPRSS2 which have been found to be important in tissue homeostasis, infection, other diseases, and in cancer, including tumor progression and metastasis.

One member of the TTSP enzyme class, matriptase (matriptase-1, MT-SP1, TADG-15, CAP3, epithin, and ST14), is a trypsin-like serine protease expressed by cells of epithelial origin and overexpressed in a wide variety of human cancers. Unlike most proteases, which are either secreted from or retained in the cell, matriptase is located on the cell surface and hence an attractive therapeutic target for a variety of therapies, including vaccines, monoclonal antibodies and small molecule compounds. Inhibition of matriptase results in concomitant inhibition of the processing and activation of multiple potential substrates important in cancer and tumor progression, including protease active receptor-2 (PAR2) and two other crucial mediators of tumorigenesis, hepatocyte growth factor (HGF) and the urokinase-type plasminogen activator (uPA).

Hepsin is another member of the type II transmembrane serine protease family. Hepsin has been reported to play a role in cancer cell growth and is known to be widely expressed with noticeably high levels in the liver and kidney as well as in cancer cells such as ovarian, breast, renal, colon, gastric and prostate. Like matriptase inhibition of hepsin results in concomitant inhibition of the processing and Activiation of multiple potential substrates important in cancer and tumor progression. Hepatocyte Growth Factor Activator (HGFA) is an S1 trypsin-like protease but is secreted and present in the blood, like other coagulation cascade proteases such as thrombin and Factor Xa. It has been associated with many tumor types similar to those described for matriptase and hepsin but also including hematological malignancies such as multiple myeloma.

Hepsin, matriptase and HGFA are differentially expressed and have upregulated function in numerous tumor types including multiple myeloma, breast, prostate, lung (and other thoracic), colon, gastric, ovarian, testicular, liver, bladder, kidney, glioblastoma and pancreatic. These proteases cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active two-chain heterodimeric forms. Active two-chain HGF and MSP are activating ligands for the receptor tyrosine kinases (RTKs), c-MET and RON, respectively.

Increased activity of hepsin, matriptase, and/or HGFA, resulting from either overexpression or upregulation of these proteases and/or downregulation of their endogenous serine protease inhibitors (serpins), HAI-1 (SPINT1), HAI-2 (SPINT2), and protein C inhibitor (PCI), has been demonstrated in tumor types driven by c-MET and/or RON receptor tyrosine kinase (RTK) pathway signaling. This increased protease function has been clearly associated with the development and elevation of metastatic cancer phenotypes, and direct inhibition of this protease activity through genetic ablation or with small molecule or antibody inhibitors has been demonstrated to reduce this metastatic potential in multiple tumor types. The biological reason for the redundancy of activation by these three different proteases and the tight regulation by serpins in cancer is not yet understood. Furthermore, since HGF/c-MET and MSP/RON signaling are necessary for development and normal cell physiology, selective inhibitors of each protease involved in individual tumors need to be identified when developing as therapeutics in order to understand and limit potential toxicities.

Matriptase inhibitors are of high therapeutic importance, but development has been a challenging task. To date, a number of small molecule inhibitors and inhibitory antibodies have been reported. See, for example, Enyedy et al.,2001, 44, 1349-1355; Steinmetzer et al., J. Med. Chem., 2006, 49: 4116-4126, and Farady et al,2007, 369: 1041-1051. Also, a series of inhibitors was recently described by Marsault et al., ACS Med Chem. Lett., 2012, 3: 530-534. Inhibitory antibodies have also been developed against matriptase.

As compared to matriptase, inhibitory antibodies have also been reported for HGFA and hepsin but relatively few inhibitors are known for either hepsin or HGFA. Small molecule hepsin inhibitors were discovered through high-throughput screening (Chevillet, J. R., et al.2008, 7, 3343) but the reported HGFA inhibitors are the non-selective serine protease inhibitors, Nafamostat and Leupeptin (Shimomura, T., et al.,1992, 8, 219). Various small molecule inhibitors of HGFA, hepsin, and matriptase are described in U.S. Patent Application Publication 2018/0066015, which is hereby incorporated by reference herein.

Although progress has been made toward the development of inhibitors of matriptase, hepsin, and HGFA, there remains a need for small molecular weight inhibitors that are both potent and selective for one or more of more of these enzymes. Such compounds have significant therapeutic value, in particular for the treatment of cancer and other conditions but most importantly those diseases involving the survival, migration, abnormal cell differentiation and proliferation of tumor cells leading to metastasis. Compounds having improved selectivity, solubility, metabolic stability, half-life, and oral bioavailability are particularly desirable.

Like matriptase and hepsin, TMPRSS2 is a TTSP and has been shown to be essential for host-cell viral entry and replication of SARS-CoV-2, SARS-CoV and other coronaviruses MERS-CoV and influenza. SARS-CoV-2 cell entry involves binding to the host (human) cell receptor ACE2. The binding of SARS-CoV-2 to ACE2 requires proteolytic priming of the Spike protein by TMPRSS2 (host (human) cell protease), suggesting that TMPRSS2 inhibitors would be effective therapeutics for COVID-19 by blocking the adherence, invasion, and replication of coronaviruses. Accordingly, TMPRSS2-expressing human lung epithelial Calu-3 cells which express both TMPRSS2 and ACE2 are highly susceptible to SARS-CoV-2 infection and other infections caused by other coronaviruses and influenza viruses which also utilize this same mechanism.

Aside from its role in SARS-CoV-2 infection, TMPRSS2 plays a role in prostate cancer (and possibly other cancers) progression and metastasis. This has been established through its ability to activate hepatocyte growth factor (HGF) (as discussed above, the sole ligand for MET receptor kinase), via proteolytic processing of pro-HGF. Thus, TMPRSS2 shares pro-hepatocyte growth factor (pro-HGF) as a protein substrate with HGFA, hepsin and matriptase. Accordingly, there remains a high unmet need for inhibitors of TMPRSS2.

Human kallikrein-related peptidases (KLKs) are a large family of S1 trypsin-like serine proteinases which are expressed in a variety of tissues such as prostate, ovary, breast, testes, brain, and skin. Although their physiological functions have been only partly elucidated, many of the KLKs appear to be useful prognostic cancer markers, showing distinct correlations between their expression levels and different stages of cancer. Of the fifteen KLKs, KLK5 is critical since it plays a role in activating many others in the family. In addition, KLK5 has been shown to activate pro-HGFA to active HGFA in cancer. Furthermore, KLK5 is known to be important in certain immune system and inflammatory conditions including allergic disorders. Accordingly, there remains a need for inhibitors of KLK5.

Generally, the present invention relates to compounds that are useful for inhibiting one or more proteases including various serine proteases not limited to Hepatocyte Growth Factor Activator (HGFA), matriptase, hepsin, thrombin, factor Xa, TMPRSS2, KLK5 and cysteine proteases including trypsin-like cysteine proteases (e.g. Cathepsin B) along with various methods of use for these compounds. In various aspects, the present invention is directed to compounds of Formula (I), salts thereof, and stereoisomers thereof:

Further aspects of the present invention relate to cyclic compounds of Formulas (IIA)-(IIH), salts thereof, and stereoisomers thereof:

wherein:

Further aspects of the present invention relate to compounds of Formula (IIIA), (IIIB), or (IIIC), a salt thereof, or a stereoisomer thereof:

wherein:

The present invention further relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one compound as described herein.

The present invention also relates to various methods of use including a method of inhibiting one or more trypsin-like S1 serine proteases (e.g., matriptase, hepsin, or HGFA) cysteine proteases including trypsin-like cysteine proteases (e.g. Cathepsin B) comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound as described herein. Another method includes a method of inhibiting HGF/MET oncogenic signaling comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound as described herein.

Other methods include a method of inhibiting carcinoma progression and/or metastasis and a method of treating a malignancy, a pre-malignant condition, or cancer comprising administering to the subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound as described herein.

Further aspects include methods of treating or preventing a viral infection in a subject comprising administering to the subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of (a) Formula (I), (IIA)-(IIH), or (IIIA)-(IIIC); (b) a polypeptide-based inhibitor; and/or (c) a benzamidine-based inhibitor. Certain aspects relate to methods of inhibiting TMPRSS2 and/or matriptase in an organism comprising administering to the organism a composition comprising an effective amount of at least one compound of (a) Formula (I), (IIA)-(IIH), or (IIIA)-(IIIC); (b) a polypeptide-based inhibitor; and/or (c) a benzamidine-based inhibitor.

Still other aspects of the invention relate to methods of treating or preventing a condition at least in part associated with KLK5 in a subject comprising administering to the subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of (a) (I), (IIA)-(IIH), or (IIIA)-(IIIC); (b) a polypeptide-based inhibitor; and/or (c) a benzamidine-based inhibitor. Some aspects also relate to methods of inhibiting KLK5 in an organism comprising administering to the organism a composition comprising an effective amount of at least one compound of (a) Formula (I), (IIA)-(IIH), or (IIIA)-(IIIC); (b) a polypeptide-based inhibitor; and/or (c) a benzamidine-based inhibitor.

Other objects and features will be in part apparent and in part pointed out hereinafter.

The present invention generally relates to compounds that are useful for inhibiting one or more various proteases including serine proteases such as Hepatocyte Growth Factor Activator (HGFA), matriptase, and hepsin as well as cysteine proteases including trypsin-like cysteine proteases (e.g. Cathepsin B). The compounds are also useful as inhibitors of KLK5 and TMPRSS2 (a necessary signal molecule for cellular entry of SARS-CoV-2). The present invention also relates to various methods of using the inhibitor compounds to treat or prevent viral infections, including those caused by coronaviruses and influenza, conditions associated with upregulated KLK5, various malignancies, pre-malignant conditions, or cancer. These methods include administering an effective amount of an inhibitor to a subject in need thereof.

Compounds of Formula (I)

In accordance with the present invention, one class of compounds useful for inhibiting one or more serine proteases includes compounds of Formula (I), salts thereof, and stereoisomers thereof:

wherein:

In various embodiments, K is a substituted or unsubstituted piperidine ring. For example, in some embodiments, K can be selected from the group consisting of:

wherein Ris hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted aralkyl; Ris hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aralkyl (e.g. benzyl), —C(O)R, or —SOOR; Ris hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle or substituted or unsubstituted aralkyl; and Ris hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted aralkyl.

In certain embodiments, Ris hydrogen, C-Calkyl, halo-substituted C-Calkyl, aryl, alkyl-substituted aryl, halo-substituted aryl, nitro-substituted aryl, alkyl-substituted aralkyl, halo-substituted aralkyl, or nitro-substituted aralkyl. For example, in some embodiments Ris hydrogen, methyl, ethyl, propyl, butyl, phenyl, benzyl, or phenethyl.

In various embodiments, Ris hydrogen, C-Calkyl, halo-substituted C-Calkyl, aryl, alkyl-substituted aryl, halo-substituted aryl, nitro-substituted aryl, alkyl-substituted aralkyl, halo-substituted aralkyl, or nitro-substituted aralkyl. For example, in some embodiments, Ris hydrogen, methyl, or ethyl.

In various embodiments, Ris hydrogen, C-Calkyl, halo-substituted C-Calkyl, aryl, aryl substituted with at least one group selected from the group consisting of alkyl, alkoxy, halo, nitro and combinations thereof. In some embodiments, Ris hydrogen, methyl, ethyl, propyl, butyl, phenyl, phenyl aryl substituted with at least one group selected from the group consisting of alkyl, alkoxy, halo, nitro and combinations thereof.

In various embodiments, K is selected from the group consisting of:

In various embodiments, L is —O—.

In various embodiments, Pis a side chain of Leu, Cha, hLeu, Nle, NptGly, hTyr, Orn, Thr, Asn, Nva, IgI, Phe, hPhe, Phe(3,4-F2), Phe(3-Cl), Phe(4-F), Phe(3-F), Glu(Bzl), Trp, Bta, hCha, hArg, Arg(Z), Lys(2-ClZ), Chg, or hTyr(Me). In some embodiments, Pis a side chain of Leu or Cha.

In certain embodiments, Rand Rare each independently hydrogen, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroarylalkyl. For example, in certain embodiments, Rand Rare each independently hydrogen, C-Calkyl, C-Ccycloalkyl, phenyl or benzyl.

In certain embodiments, Ris hydrogen, alkyl, or a residue of an amino acid, or Rand Rcan form a piperazine or piperidine ring

In various embodiments, each Ris independently hydrogen, alkyl, or the Rmoieties can form a ring.

In various embodiments, Ris aryl.